Permissiveness of Guinea Pig Alveolar Macrophage Subpopulations to Acute Respiratory Syncytial Virus Infection In Vitro: Materials and Methods

In: Respiratory

16 Jul 2014

Virus
The Long strain A of human RSV was obtained (American Type Culture Collection [ATCC]; Manassas, VA). RSV was propagated on HEp-2 cell monolayers (ATCC) in minimal essential medium (MEM) supplemented with 2% heat-inactivated fetal bovine serum (FBS) and 50 μg/mL gentamicin (Gibco; Grand Island, NY). When > 75% of cells exhibited cytopathic effect (formation of syncytia, cell rounding, and detachment), the culture flasks were shaken with autoclaved 3-mm glass beads and the resulting cell suspension was sonicated on ice for 1 min at the maximum output from a sonicator (Vibra Cell; Sonics & Materials Inc; Danbury, CT). Cellular debris was removed by centrifugation at 1,500 X g and 4°C for 15 min and the cleared supernatant containing virus was kept at — 70°C and used for infection immediately after determining the titer by plaque assay.
Animals
Juvenile female Cam Hartley guinea pigs, 22 to 29 days old (250 to 300 g body weight), were purchased (Charles River Laboratories; Montreal, QC, Canada) and were housed as previously described, in conditions of plastic cages containing corn cob bedding, access to guinea pig chow (Purina; Ralston Purina Company; St. Louis, MO), alfalfa hay cubes and water, and 12-h alternating light-dark cycles. Canadian health and care mall read Animals were maintained in accordance with standards of the Canadian Council on Animal Care.
BAL, Blood Collection, and Cell Processing
Animals were anesthetized by intraperitoneal administration of pentobarbital (Euthanyl; MTC Pharmaceuticals; Cambridge, ON, Canada), exsanguinated by cardiac puncture, and 5 mL of blood was collected in EDTA-containing tubes. Blood mononuclear cells were isolated from whole blood by standard methods using Ficoll-paque density gradient centrifugation (Pharmacia Biotech; Uppsala, Sweden). BAL was performed in situ by instilling, intratracheally, 5 to 10 mL aliquots of sterile, nonpy-rogenic normal saline solution, prewarmed at 37°C (total volume: 100 mL). After each instillation, the fractions were gently aspirated and the fluids were pooled in 50-mL centrifuge tubes and kept on ice until processed. Typically, the percent recovery of lavage fluid by this method was > 95% and no blood contamination occurred during the lavage procedure. BAL cells were sedimented by centrifugation at 500 X g for 10 min at 4°C, and counted in an automated cell counter (Coulter Electronics Inc; Hialeah, FL). More than 85% of total cells recovered by lavage were AMs (mean ± SD: 87.8 ± 2.6%). Cell viability, as determined by trypan blue dye exclusion, was > 95%.


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